J. Agric. Food Chem. 2002, 50, 1833−1839
1833
Study of Catechin and Xanthine Tea Profiles as Geographical Tracers PEDRO L. FERNAÄ NDEZ, FERNANDO PABLOS, MARIÄA J. MARTIÄN,* A. GUSTAVO GONZAÄ LEZ
AND
Department of Analytical Chemistry, Faculty of Chemistry, University of Seville, 41012 Seville, Spain
The contents of gallic acid, epigallocatechin gallate, epigallocatechin, epicatechin, epicatechin gallate, catechin, caffeine, teophylline, and theobromine were determined in a set of 45 tea samples, including fermented (black and red) and nonfermented (green) teas of different geographical origins (i.e., China, Japan, Kenya, Sri Lanka, and India). A reversed-phase high performance liquid chromatographic method with gradient elution and photometric detection at 275 nm was used to carry out the analysis. Before the HPLC determination, an extraction step was developed using a mixture of acetonitrile and water (60:40, v/v). Pattern recognition techniques involving principal component analysis (PCA) and linear discriminant analysis (LDA) were applied to differentiate the tea samples according to their geographical origins. Catechins, gallic acid, and tea alkaloids are adequate chemical descriptors to distinguish between fermented and nonfermented tea samples cultivated in different geographical areas. KEYWORDS: Tea; polyphenols; catechins; xanthines; HPLC; chemometrics; pattern recognition
INTRODUCTION
Tea is a beverage consumed in the entire world because of its attractive aroma, taste, and healthy effects. Tea plants (Camellia sinensis) are widely cultivated in over 30 countries and are significant in their economies (1). Tea-producing areas are located mainly in Southeast Asia, including China, India, Japan, Taiwan, Sri Lanka, and Indonesia, and in central African countries such as Kenya. Nowadays, hundreds of teas are produced. Commercial teas can be generally classified into three major categories: the nonfermented green teas, the partially fermented oolong and paochong teas, and the fully fermented black and pu-erh (red) teas (2). Many factors can constitute important influences on the composition of tea, such as species, season, age of the leaves (plucking position), climate, and horticultural conditions (soil, water, minerals, fertilizers, etc.) (3). Catechins, together with phenolic acids such as gallic acid (GA), are a group of polyphenols that constitute up to 30% of the dry weight of the tea leaf and are important factors in the taste of tea. These compounds are the most biologically active group of tea components, especially certain catechins. The major tea catechins are epigallocatechin gallate, epigallocatechin, epicatechin, and catechin (2). Some of the biological functions of tea polyphenols are antioxidative activity (4), and antihypertensive (5) and anticarcinogenic effects (6, 7). Caffeine, theophylline, and theobromine are the main methylxanthines constituting the tea alkaloids, being important factors in the quality of teas. Tea * To whom correspondence should be addressed (telephone 34-954557173; fax 34-954-557168; E-mail
[email protected]).
also contains minerals and trace elements such as K, Mn, Cr, Ni, and Zn, which are essential to human health (8). Several studies determining the tea catechins and tea alkaloids, separately (9, 10) and simultaneously (2, 11), have been carried out. Though several authors propose capillary electrophoresis as the technique to be used (12-14), the analytical method commonly used for the determination of these compounds is high-performance liquid chromatography (HPLC), which currently constitutes the most useful approach for routine analysis. Many works have been reported including HPLC determinations of these tea polyphenols with isocratic (2, 15) and gradient elution (11, 16-19). As it has already been mentioned, climate and agricultural practices, including soil, water, and fertilizers, can be of great influence on the composition of teas. Thus, teas cultivated in different geographical areas will present significant differences in their chemical compositions. Metals have been found to be adequate descriptors to distinguish teas of different geographical origins (20, 21). In this paper, the contents of five catechins (catechin (C), epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG)), one phenolic acid (gallic acid (GA)), and three alkaloids (theobromine (TB), theophylline (TP), and caffeine (CAF)) (Figure 1) have been analyzed in teas cultivated in different areas (China, Japan, Kenya, Sri Lanka, and India). These parameters have been considered as chemical descriptors to differentiate teas according to their geographical origins. Pattern recognition techniques,
10.1021/jf0114435 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/03/2002
1834
J. Agric. Food Chem., Vol. 50, No. 7, 2002
Ferna´ndez et al.
Figure 1. Structures of gallic acid, catechins, and tea alkaloids.
such as principal component analysis (PCA) and linear discriminant analysis (LDA), were applied for classification purposes. MATERIALS AND METHODS Apparatus. A Merck Hitachi (Darmstadt, Germany) HPLC chromatograph, equipped with a L-7100 pump, a Rheodyne (Cotati, CA) 7725i injection valve with a 20-µL sample loop, and a diode array detector L-7455 operating at 275 nm was used for the determinations. All the modules were controlled by a personal computer equipped with a Merck Hitachi D-7000 interface and HPLC System Manager software (Merck Hitachi). A 25 cm × 4 mm LichroCART RP-18 5-µm column (Merck) was used for the separation. Reagents and Standard Solutions. Formic acid was obtained from Panreac, Barcelona, Spain. Acetonitrile (Romil, Cambridge, UK) was of HPLC grade. Milli-Q (Millipore, Bedford, MA) treated water with a resistivity higher than 18 MΩ cm was used throughout. Gallic acid, (+)-catechin, and (-)-epicatechin were purchased from Fluka (Buchs, Switzerland). Theobromine, theophillyne, (-)-epigallocatechin, (-)epicatechin gallate, and (-)-epigallocatechin gallate were acquired from Sigma (Steinheim, Germany). These reagents were stored at -20 °C. Caffeine was obtained from Merck. Stock standard solutions (200 µg mL-1) were prepared in acetonitrile and stored at 4 °C. Working standard solutions were prepared weekly from the stock solutions by dilution with acetonitrile. Samples. A set of 45 commercial tea samples obtained from herbalists and specialized markets was selected for the analysis. Of the set of 45, 13 samples were nonfermented teas (green) and 32 were fermented teas (black and red). In all cases, the countries of origin were known, being China, Japan, Sri Lanka, Kenya, and India. In the case of teas from China and India, it was also known from which region of the country the teas were cultivated. In relation to the technology used in the manufacture of black teas, it was not possible to know which were CTC or orthodox. Table 1 shows a short description of the samples and the corresponding code assigned to each one. Sample Preparation. The tea samples were extracted according to the following procedure: 0.5 g of tea sample was extracted with 100 mL of acetonitrile/water (60:40, v/v) at room temperature for 1 h with constant stirring. The extract was filtered and diluted to volume in a 100-mL calibrated flask. Aliquots of 10 mL of this solution were transferred to a 25-mL volumetric flask rising with ultrapure water.
Portions of this solution were filtered through a disposable 0.45-µm filter unit into a vial and injected into the HPLC system. This extraction procedure was carried out at room temperature to prevent possible degradation of the catechins (22). To assess the rudegeness of the extraction procedure, some samples were extracted within a weight range of 0.35-0.75 g. No significant differences were found in the determined contents of catechins and xanthines, regardless of the sample size taken from the extraction. As the results have been expressed on a dry basis, the moisture of the tea samples was determined before the analysis. HPLC Method. The analytical determinations of gallic acid, theobromine, theophylline, catechin, caffeine, epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate were carried out by means of high-performance liquid chromatography using a twosolvent gradient elution (11). The solvents compositions were (A) water/ acetonitrile/formic acid (94.7:4.3:1 v/v) and (B) water/acetonitrile/formic acid (49.5:49.5:1 v/v). The mobile phase composition started at 90% solvent A and 10% solvent B, was increased linearly to 30% solvent B in 10 min, followed by a linear increase of solvent B to 80% in 5 min, and the final conditions being held for an additional 3 min. The flow rate was always 1 mL min-1. To check the performance of the method the selectivity, linearity, detection and quantification limits, accuracy, and precision were evaluated. Resolutions of the peaks were calculated, and were higher than 1.5 in all the cases. The limit of detection (LOD) and limit of quantification (LOQ) were calculated as the concentration giving a signal equal to 3 and 10 times, respectively, of the signal/noise ratio (23). The accuracy of the method was evaluated from recovery assays, preparing spiked tea samples in triplicate at several levels of concentration. The average recoveries were calculated according to Cuadros et al. (24). Table 2 summarizes the linear range, LOD, LOQ, and recovery values. The precision of the method was evaluated by carrying out eight replicate analysis of a standard solution on different days. The obtained relative standard deviation was always
